Humoral immunity plays a major role in the protection mediated by most vaccines, including the smallpox vaccine (vaccinia virus, W). Memory B cells are a central component of humoral immunity, and yet little is known about their characteristics, their longevity in humans, or their protective value. In the interest of better understanding the role of human memory B cells in protection against disease, we have developed an assay to quantitate antigen-specific memory B cells in human blood. Using this assay we recently demonstrated that smallpox vaccine specific memory B cells last for greater than 50 years in immunized individuals (Crotty et al, Jl 2003). Immune memory after smallpox vaccination is a valuable benchmark for understanding the longevity of B cell memory in the absence of re-exposure to antigen. These persisting human memory B cells were functional and able to mount an anamnestic antibody response upon re-vaccination. Additionally, W-specific CD4+ T cells and antibodies were detected decades after vaccination. This proposal follows up that study and explores the functional phenotypes of antigen-specific memory B cells, the interrelationship of memory B cell and antibody levels, and the characteristics of protective memory B cell and antibody responses. These studies will further elucidate fundamental principles of human antiviral humoral immunity. Our specific aims are: (1) Are there distinct functional subsets of memory B cells? (2) What are the interrelationships between the components of long term humoral immunity? (3) Is the memory B cell recall response relevant for protection? (4) Are antibodies directed against viral immune evasion virulence factors (lEVFs) valuable components of the host immune response?